CYTOLYTIC T LYMPHOCYTE HELPER FACTOR

溶细胞T淋巴细胞辅助因子

• 批准号: 3172816
• 负责人: DAVID P FAN
• 金额: $ 19.35万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3172816
• 关键词: Anura; T lymphocyte; antibody dependent killer cell; complementary DNA; cytokine; flow cytometry; genetic manipulation; high performance liquid chromatography; histocompatibility antigens; laboratory mouse; laboratory rat; leukocyte activation /transformation; molecular cloning; monoclonal antibody; nucleic acid probes; nucleic acid sequence; protein sequence; radiotracer; receptor; tissue /cell culture; transposon /insertion element; tumor antigens

We have developed an assay for cytokines needed for the in vitro generation of primary mouse cytolytic T lymphocytes (CTL) against alloantigens on tumor cells. One of the factors which functions in this assay is called RDCHF. We have recently purified RDCHF to a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The cytokine could be eluted from the gel with retention of biological activity. From a preliminary sequence of the first 10 amino acids, RDCHF is different from all sequenced cytokines. We have also demonstrated that at least one RDCHF clone exists in a cDNA library we have made from RDM4 cells producing RDCHF. We have begun to subdivide the library to isolate cDNA clones. We propose to refine the RDCHF purification to achieve a reproducible technique for obtaining pure material. We will bind radiolabeled RDCHF to cells from established lines or from preparations of crude cells separated by fluorescent activated cell sorter (FACS). In this way, we will identify the immediate target cells of the factor. We will also bind radiolabeled RDCHF covalently to its receptor(s) on target cells to study the nature of the receptor(s). We also plan to complete the isolation of cDNA clones to RDCHF. We will generate transient expression vectors to express the cytokine in large amounts in the COS cell line to produce large amounts of protein for biological studies. We will use the cDNA clone to isolate genomic clones to study the structure of the gene coding for the factor. We will perform hybridization studies to identify the cells making RDCHF and to study the expression of the RDCHF gene. We will also attempt to isolate a homologous gene from the human genome. Once we can make large amounts of purified RDCHF, we will attempt to make monoclonal antibodies to this cytokine both for biological studies and for simplification for factor purification.

我们开发了一种体外细胞因子测定方法 原代小鼠溶细胞性 T 淋巴细胞 (CTL) 的产生 对抗肿瘤细胞上的同种异体抗原。 其中的因素之一 该测定中的功能称为 RDCHF。 我们最近用钠将 RDCHF 纯化为单条带 十二烷基硫酸盐-聚丙烯酰胺凝胶电泳（SDS-PAGE）。 细胞因子可以从凝胶中洗脱并保留 生物活性。 从前 10 个的初步序列来看 氨基酸，RDCHF 与所有已测序的细胞因子不同。 我们还证明了至少存在一个 RDCHF 克隆 在我们用 RDM4 细胞制作的 cDNA 文库中，产生 RDCHF。 我们已经开始对文库进行细分来分离cDNA 克隆。 我们建议改进 RDCHF 纯化以实现 获得纯材料的可重复技术。 我们将绑定 将放射性标记的 RDCHF 标记到来自已建立细胞系或来自 荧光激活细胞分离的粗细胞制剂 分选机（FACS）。 这样我们就能确定当前的目标 因子的细胞。 我们还将结合放射性标记的 RDCHF 与靶细胞上的受体共价结合以研究其性质 受体。 我们还计划完成 RDCHF cDNA 克隆的分离。 我们将生成瞬时表达向量来表达 COS细胞系中大量细胞因子产生大量 用于生物学研究的蛋白质量。 我们将使用 cDNA 克隆分离基因组克隆以研究基因结构 因子的编码。 我们将进行杂交研究 鉴定产生 RDCHF 的细胞并研究 RDCHF 基因。 我们还将尝试分离出同源的 来自人类基因组的基因。 一旦我们能够生产大量纯化的 RDCHF，我们将 尝试制备针对该细胞因子的单克隆抗体 生物学研究和因子纯化的简化。